Certain naturally occurring RNA molecules undergo self-catalyzed cleavage (Kruger et al., 1982). The best studied examples occur in plants that are infected with viroids (Hutchins et al., 1986), virusoids (Forster & Symons, 1987a), or satellite viruses (Prody et al., 1986). The genomic RNAs of these infectious agents are reproduced by a rolling circle mechanism that yields multimeric replication intermediates that undergo self-cleavage to produce monomeric genomes. The ability to self-cleave is imparted by distal consensus sequences that interact to form a highly structured RNA configuration (in the form of a "hammerhead") prior to cleavage (Forster & Symons, 1987a, Forster & Symons, 1987b). Although self-cleavage is entirely intramolecular, a careful analysis of the sequences and structures involved led Uhlenbeck to the realization that a synthetic RNA could be constructed that would interact with a second RNA (the "substrate" strand) to form the hammerhead configuration, resulting in the cleavage of the substrate at a specific site (Uhlenbeck, 1987). He demonstrated that these synthetic RNAs act enzymatically.
Uhlenbeck's experiments raised the prospect that RNA enzymes ("ribozymes") could be constructed that would cleave a preselected sequence in any RNA. However, in his scheme some of the consensus sequences which were required to form the active hammerhead configuration were supplied by the substrate strand, severely limiting the number of natural RNAs that could serve as substrates. Haseloff and Gerlach markedly improved Uhlenbeck's design by including all the required consensus sequences in the ribozyme (Haseloff & Gerlach, 1988). Their ribozyme works by first hybridizing to a particular site in the substrate RNA and then catalyzing the cleavage of the substrate at that site. They disclose that sites containing the trinucleotide GUC, and perhaps GUU or GUA, are cleaved, provided that the structures present in the substrate RNA do not prevent the binding of the ribozyme to the site. FIG. 1A shows a typical Haseloff-Gerlach ribozyme hybridized to a substrate strand. The arrow indicates the site in the substrate where cleavage will occur. This site is immediately adjacent to the GUC sequence.
Haseloff-Gerlach ribozymes possess two different functional regions. The first functional region is a "catalytic domain" in the middle of the ribozyme (FIG. 1B), which contains the consensus sequences that confer the ability to cleave the substrate. The catalytic domain is example shown is 22 nucleotides-long. This region is common to Haseloff-Gerlach ribozymes. The second functional region consists of sequences on both sides of the catalytic domain. These sequences are chosen to be complementary to the sequences surrounding the cleavage site in the substrate (FIG. 1C). They confer upon the ribozyme the ability to interact specifically with a preselected cleavage site. Because the combined length of these complementary sequences is typically between 12 and 16 nucleotides, the Haseloff-Gerlach ribozymes are highly specific.
Experiments that tested the activity of Haseloff-Gerlach ribozymes in vivo have been disappointing. The expression of targeted gene products was not eliminated, but only reduced (Cameron & Jennings, 1989), and extremely high levels of ribozyme were required to destroy the intended substrate RNA (Cotten et al., 1989). Furthermore, our own in vitro studies showed that the optimal conditions for ribozyme activity (60 degrees Celsius in the presence of 40 mM magnesium chloride) are quite different from the conditions present in most eukaryotic cells. Naturally occuring hammerhead configurations are optimal only for cleavage within the same RNA (cleavage in cis). Moreover, in plant cells (where hammerheads normally function), cleavage might be aided by accessory proteins. The Haseloff-Gerlach ribozymes, on the other hand, are designed to cleave another RNA (cleavage in trans), without the benefit of cellular proteins. Furthermore, the Haseloff-Gerlach ribozymes might be particularly sensitive to cellular nucleases. It is thus not surprising that artificial ribozymes function less than optimally in vivo. The utility of therapeutic ribozymes will depend on their ability to function efficiently in a complex cellular milieu (Sarver et al., 1990). Thus, it is clear from our own work and from the work of other laboratories that the efficiency of ribozymes as currently designed, either Haseloff-Gerlach or other, is too low for them to serve as commercially effective therapeutic agents. A purpose of the invention described herein is to design and screen ribozymes that will efficiently cleave target RNA.